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2011 International Conference on Food Engineering and Biotechnology
IPCBEE vol.9 (2011) © (2011)IACSIT Press, Singapoore
Challenge to healthcare: Multidrug resistance in Klebsiella
pneumoniae
Archana Singh Sikarwar1 and Harsh Vardhan Batra2
1
Faculty of Medicine and Health Science, International Medical University,
Bukit Jalil,Kuala Lumpur, Malaysia
2
Scientist E, Defense Research Development and Establishment,
Gwalior, India
Abstract. Klebsiella pneumoniae has been associated with different types of infections and one of the
important aspects of Klebsiella associated infection is the emergence of multi-drug resistant strains
particularly those involved in nosocomial diseases. For our studies we have collected fifty – nine clinical
isolates, from different places of India as this study was conducted in India. Morphologically resembled
Klebsiella were recovered from respiratory, UTI and pus cases. Out of fifty- nine clinical isolates, twenty
were found positive for K.pneumoniae at biochemical characterization. These clinical isolates of
K.pneumoniae were further tested for antimicrobial sensitivity and most of them were found to be multidrug
resistant. All confirmed K.pneumoniae isolates were resistant to carbenicillin and one among them recovered
from sputum sample of a pneumonic patient was resistant to all the antimicrobial agents tested except
exhibiting a partial susceptibility to amikacin. Klebsiella pneumoniae strains from clinical cases in our study
were found highly susceptible to quinolones, aminoglycoside, amykacin and gentamycin. At the same time
over 60% strains were resistant to chloramphenicol and tetracycline. We also found that 28 to 76% of them
were resistant to cephalosporins (ceftizoxime and cefotaxime).
Keywords: Klebsiella pneumoniae, Antimicrobial sensitivity, multidrug resistance
1. Introduction
Klebsiella are ubiquitously present and reported worldwide. In recent years, Klebsiellae have become
important pathogens in nosocomial infections [1]. The importance of Klebsiella species in the ever
increasing number of gram negative aerobic bacillary nosocomial infections in the United States [2] and
India [3] has been well documented. Epidemic and endemic nosocomial infections caused by Klebsiella
species are leading causes of morbidity and mortality [4]. In addition to being the primary cause of
respiratory tract infections like pneumonia, rhinoscleroma, ozaena, sinusitis and otitis, it also causes
infections of the alimentary tract like enteritis, appendicitis and cholycystitis. They are frequently associated
with the infections of urinary tract, genital tract, and the eyes. Statistical data and evidences from researches
prove that multidrug resistance bacteria are emerging worldwide which is a big challenge to healthcare.
Multidrug resistant bacteria cause serious nosocomial and community acquired infections that are hard to
eradicate using available antibiotics. Moreover, extensive use of broad-spectrum antibiotics in hospitalized
patients has led to both increased carriage of Klebsiella and the development of multidrug-resistant strains
that produce extended–spectrum beta-lactamase (ESBL). Epidemic strains of cephalosporin resistant
K.pneumoniae have been associated with increased morbidity and mortality in hospitalized patients [5].
Since 1983, nosocomial outbreaks of ESBL producing K.pneumoniae infections in Europe [6], the United
States and South America have been described [7,8]. Between 1990 and 1992, 5% of K.pneumoniae clinical
isolates produced ESBLs [9]. In France, 10 to 30 % of K.pneumoniae strains are reported to produce plasmid
mediated ESBLs of the TEM or SHV families [7]. We have started our studies with following aims and
objectives:
•
To analyze the severity of multidrug resistance of Klebsiella pneumoniae in India
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•
Challenges faced by healthcare community in India during treatment of multi drug resistant
K.pneumonic patients.to overcome this problem.
•
To come up with some suggestions to overcome multidrug resistant problem.
2. Materials and Methods
We have collected standard and clinical isolates of K.pneumoniae from different places of India
and utilized them for their antibiotic resistant ability.
2.1. Bacterial strains:
Standard culture of K.pneumoniae strain 3296 was collected from Yamaguchi University, Japan whereas
total fifty nine clinical isolates were collected from different places of India. Out of all, fifty - four clinical
isolates of Klebsiella species were recovered from pus, urine and sputum samples from Armed Force
Medical College, Pune, India, four samples from pneumonic patients and one UTI sample from Patel
Chest Hospital, New Delhi, India.
2.2. Biochemical characterization:
All the clinical isolates were examined morphologically for colony characteristics on agar media. Those
exhibiting mucoid colonies were processed for biochemical testing. Biochemical test employed were urease
production, citrate utilization and fermentation of sugars. Sugar fermentation tests performed were sucrose,
glucose, mannitol, lactose, adonitol, dulcitol, melibiose and esculin. Indole test and H2S production on TSI
agar, oxidase, catalase and nitrate were also carried out. Besides these tests, motility and growth of organism
in potassium cyanide were also checked. For biochemical tests standard procedures were used [10].
2.3. Antibiotic sensitivity testing:
Standard and positive clinical samples of K.pneumoniae were grown on nutrient agar. After 24 hours
incubation at 37oC, fresh colonies of K.pneumoniae were grown in a tube containing 5mL of a tryptic-soy
broth followed by overnight incubation at 37°C until it achieves the turbidity. Next day, dipped a sterile nontoxic swab into the broth and rotated the swab several times, pressing firmly on the inside wall of the tube
above the fluid level to remove excess inoculum from the swab. Organism was inoculated over the dried
surface of a Muller-Hinton agar plate by streaking the swab over the entire sterile agar surface three times,
rotating the plate 60oC each time . We have placed the antibiotic discs evenly on the surface of the agar plate
by using a sterile forceps then plates were placed in incubator at 37°C. After 24 hours of incubation, we
examined each plate and reported the organism to be susceptible, moderately susceptible (intermediate), or
resistant for antibiotics.
3. Results
The standard and clinical isolates of Klebsiella were examined by battery of biochemical tests. Standard
K.pneumoniae cultures showed positive reactions for urease production, citrate utilization, catalase reaction
and fermentation of sugars like glucose, lactose, sucrose, mannitol, adonitol, melibiose and esculin.
Organisms showed negative test for indole production. There was no H2S production on Triple Sugar Iron
agar but growth of organism was seen in KCN. All fifty-nine clinical isolates exhibiting colonies similar to
Klebsiella species were tested biochemically. Thirty-six clinical isolates showed typical common
biochemical reaction pattern similar to the one seen with Klebsiella species, being positive to glucose,
lactose, sucrose, mannitol and negative to oxidase and indole. When tested by second battery of biochemical
reactions that included some rare sugars (adonitol, melibiose, esculin and dulcitol), twenty of them exhibited
reactions attributable to majority of K.pneumoniae sub species pneumonia strain, being positive to adonitol,
melibiose, esculin, urease and citrate. They also showed growth in KCN. The rest of the isolates had variable
reactions with these tests.
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Antibiotic sensitivity testing of twenty confirmed K.pneumoniae clinical isolates was done on Muller –
Hinton agar plates. On the basis of resistance to antibiotic, strains were categorized in three groups. One
group had strains which were susceptible (over 85%) to quinolones and aminoglycosides. The second group
had strains which were moderately susceptible (intermediate) to antiribosomal antibiotics (chloramphenicol
and tetracycline) with 62% resistant strains. The third group contained strains which were resistant to semisynthetic penicillins, ampicillin, carbenicillin (76-100%) and to co-trimoxazole (76%). Results are shown in
fig 1.
Fig 1: Antibiotic resistance pattern of Klebsiella pneumoniae isolates
4. Discussion
In vitro data showed a wide range of beta-lactams, aminoglycosides, quinolones and other antibiotics are
useful for treatment of klebsiellae infections [11, 12, and 13]. The clinical isolates of K.pneumoniae were
tested for antimicrobial sensitivity and most of them were found to be multidrug resistant. All the Klebsiella
pneumoniae isolates were resistant to carbenicillin and one among them recovered from sputum sample of a
pneumonic patient was resistant to all the antimicrobial agents tested except exhibiting a partial susceptibility
to amikacin. In such cases the disease is prone to progress to permanent debilitation or death of the patient if,
isolation and identification of the causative agent and the subsequent antimicrobial susceptibility testing is
not carried out at the early stage of the disease.
In our studies, K.pneumoniae strains from clinical cases were found highly susceptible to quinolones and
aminoglycoside, amikacin and gentamycin. At the same time over 60% strains were found resistant to
chloramphenicol and tetracycline. Twenty-eight to 76% of them were resistant to cephalosporins
(ceftizoxime and cefotaxime). Cephalosporins have been widely used as monotherapy and in combination
with aminoglycosides for the treatment of Klebsiella infection.
Plasmid encoded resistance to broad spectrum cephalosporins is becoming a widespread phenomenon in
clinical medicine. These antibiotics are inactivated by an array of different extended spectrum beta
lactamases (ESBLs) which have evolved by stepwise mutation of TEM/SHV type beta lactamases. Plasmid
encoding these enzymes has been encountered in several members of the family enterobacteriaceae, but are,
for unknown reasons, most often harboured by K.pneumoniae [14]. Epidemic and endemic nosocomial
infections caused by ESBL producing K.pneumoniae represent a persistent problem in many parts of the
world, especially in ICUs [15 and 16]. The emergence of multidrug resistant strains particularly those
involved in nosocomial diseases and the alarming rise in resistance to SHV and ESBL producing groups of
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antibiotics result in high morbidity and mortality. Early identification of agent, therefore, is important for
timely management of patients.
Klebsiella has been associated with different types of infections and one of the important aspects of
Klebsiella associated infection is the emergence of multi-drug resistant strains particularly those involved in
nosocomial diseases.The alarming rise in resistance to SHV and ESBL producing groups of antibiotics result
in high morbidity and mortality.TEM- and SHV type ESBL producing Klebsiella pneumoniae were
extensively reported worldwide after it was first identified in enterobacterial isolates from India [17]. The
high prevalence of these drug resistant strains has further necessitated the requirement of a rapid and
accurate identification system for K.pneumoniae.
In our studies, we have found that out of fifty – nine clinical isolates, twenty clinical isolates were found
positive for K.pneumoniae at biochemical characterization. When these clinical isolates of K.pneumoniae
were further tested for antimicrobial sensitivity and most of them were found to be multidrug resisitant. The
isolates were highly susceptible quinolones and the aminoglycosides. Over sixty percent strains were
resistant to chloramphenicol and tetracycline but all the isolates were resistant to carbenicillin.
5. Conclusion
Statistical data and evidences from researches prove that multi drug resistant bacteria are emerging
worldwide which causes many public health problems and challenges to healthcare. Moreover, uses of broad
spectrum antibiotics, insufficient aseptic condition and technique with inadequate control of infections
spread had aggravated this problem. Recently, WHO warned society during press release and stated that
antibiotics may lose their power to cure disease if action is not taken now against antimicrobial resistance
problem [18]. WHO also recommended six ways to overcome multi drug resistant problem, those are:
•
Committing to a comprehensive, financed national plan with lines of accountability and community
engagement;
•
Strengthening surveillance and laboratory capacity;
•
Ensuring a regular supply of good-quality medicines;
•
Regulating and promoting rational use of medicines and proper patient care;
•
Enhancing infection prevention and control in health settings; and
•
Fostering innovation, research and development
6. Acknowledgement
I gratefully acknowledge the Director of the Defense Research and Development Establishment, Gwalior,
India for encouragement and support. This work was funded by the ministry of Human Resources,
Government of India, (Graduate Aptitude Test Examination) scholarship. I would also like to thank Dr
J.Uchiyama (Yamaguchi University, Japan), Armed Force Medical College, Pune and Patel Chest Hospital,
New Delhi for providing us standard and clinical bacterial isolates.
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